Enhancing the effect of therapeutic proteins on the central nervous system

ABSTRACT

The present invention provides a polypeptide therapeutic agent, useful in enzyme replacement therapy, with increased therapeutic benefits for the central nervous system. The invention provides a method of enhancing the effect of a polypeptide or protein on the central nervous system by the attachment of a short acidic amino acid sequence. Specifically the inventors disclose the attachment of a 4-15 acidic amino acid sequence to human β-glucuronidase by construction of a fusion protein. This molecule is useful in the treatment of type VII mucopolysaccharidosis when administered to a patient.

PARENT CASE TEXT

This application claims benefit of priority to a continuation in part ofU.S. application Ser. No. 11/245,424, filed Oct. 7, 2005, and also acontinuation in part of U.S. application Ser. No. 10/864,758, filed Jun.10, 2004.

Sequence Listing

A paper copy of the sequence listing and a computer readable form of thesame sequence listing are disclosed in U.S. application Ser. No.11/245,424, filed Oct. 7, 2005 to which this application claims benefitof priority as a continuation in part, and is herein incorporated byreference.

GOVERNMENT SUPPORT CLAUSE

This work was supported by the National Institutes of Health grantnumber GM34182, and International Morquio Organization. U.S. Governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to endowing therapeutic protein agentswith increased in vivo stability and effectiveness on the centralnervous system (CNS). More specifically, the present invention relatesto endowing β-glucuronidase protein (GUS) with improved stability in theblood and enhanced ability to affect the CNS, in a therapeutic capacityby attaching a short peptide of acidic amino acids to the N-terminus ofthe protein.

2. Description of the Related Art

Lysosomal storage diseases (LSDs) are a class of forty rare geneticdisorders, each of which is caused by a deficiency in a specificlysosomal enzyme. As a consequence of the progressive accumulation ofunmetabolized macromolecules in the lysosomes of cells in varioustissues, the disease manifestations worsen over time.¹ Individualsafflicted with an LSD can suffer from mild to severe physical and/orneurological abnormalities or can die at an early age. A therapeuticparadigm for the treatment of LSDs was established with the success ofenzyme-replacement therapy (ERT) for the treatment of Gaucherdisease.^(2,3) In the case of Gaucher disease, delivery of the enzyme tothe affected cells was achieved by modifying the N-linked carbohydrateon the enzyme. This exposed core mannose residues,^(4,5) enabling theenzyme to bind to the MR, which is highly abundant on cells of thereticuloendothelial system.^(6,7) These findings led to clinicalmanagement of Gaucher disease by ERT.⁸ Over 3,500 patients have beentreated with dramatic clinical results.⁹

Meanwhile there is a problem that pharmaceutical preparations ofphysiologically active proteins like enzymes and peptide hormones aregenerally made unstable when they are administered to the body, and thusundergo relatively rapid inactivation by, e.g., enzymatic degradation.For pharmaceutical preparations of a physiologically active protein, amethod for increasing the stability of the physiologically activeprotein in the body is known which is based on coupling the proteins topolyethylene glycol.¹⁰

Sly's syndrome is an autosomal recessive, genetic lysosomal storagedisease caused by an anomaly in the gene for a lysosomal enzyme,β-glucuronidase (hereinafter referred to as GUS) ¹¹(6), and isclassified as type VII mucopolysaccharidosis (hereinafter referred to asMPS VII). In lysosomes, GUS acts as an exoglycosidase to removeglucuronic acid residues from the non-reducing termini of GAGs(glycosaminoglycans), such as dermatan sulfate (DS), heparan sulfate(HS), and chondroitin sulfate (CS). In the absence of GUS, GAGs are onlypartially degraded and accumulate in lysosomes of a variety of tissues.Progressive accumulation of undegraded GAGs in lysosomes affects thespleen, liver, kidney, cornea, brain, heart valves, and the skeletalsystem, leading to facial dysmorphism, growth retardation, systemic bonedysplasia, deafness, mental retardation, and shortened lifespan.

No effective remedy is currently available for MPS VII. Enzymereplacement therapy (ERT) has been considered to be the potential remedyfor MPS VII. Considering its rapid inactivation in the body, however,native GUS is not expected to give any satisfactory effect.

The challenge is to improve joint and brain-related pathology since mostof the enzyme-based drugs are delivered to major visceral organs likeliver and spleen and only a small amount of enzyme is delivered to boneand brain. Many lysosomal enzymes have a short half-life when injectedinto the bloodstream because of rapid clearance in the liver bycarbohydrate-recognizing receptors, particularly the mannose receptorthat is highly abundant on Kupffer cells.¹² Although a part of theenzyme reaches the bone marrow, there is no way to guarantee that theenzyme will reach the brain since the blood brain barrier presents aformidable obstacle. As a result, current ERT doses not work efficientlyon the bone and brain lesions.

The inventors have sought to address the problem of stability oftherapeutic proteins in vivo and the inability of some proteins toeffectively cross the blood brain barrier. The inventors have previouslydisclosed the use of short peptides of acidic amino acids to targetproteins to bone tissue for use in Enzyme Replacement Therapy (ERT).¹³The inventors have also disclosed the use of short peptides of acidicamino acids to improve stability of physiological active proteins in theblood.

The addition of 4-15 acidic amino acids to GUS results in an increase inmolecular weight which generally, would not be expected to increasefunctional activity of proteins to the CNS. In fact, higher molecularweigh molecules are more effectively excluded from the brain by anineffectual crossing the blood brain barrier. Similarly, an increase inthe hydrophilic nature of a molecule is also thought to excludemolecules at the blood brain barrier. The inventors have made thesurprising discovery that despite causing an apparent increase inmolecular weight and increase in hydrophilic nature, the addition of anacid amino acid leader to GUS has allowed enhanced therapeutic benefitson the brain.

SUMMARY OF THE INVENTION

An object of the present invention is a method to increase in vivostability of a physiologically active peptide or protein by the additionof a short acidic amino acid leader, and thereby increase itstherapeutic effects on the CNS for treatment of CNS related disease.

Previously, the inventors made the unexpected discovery thatN-acetylgalactosamine-6-sulfate sulfatase (GALNS), tissue-nonspecificalkaline phosphatase (TNSALP) and GUS with a short amino acidic peptide(AAA) attached to the N-terminus increased targeting and deposition ofthese enzymes to bone. They further discovered that GALNS and GUS, withthis short acidic amino acidic peptide attached possessed improved invivo stability in the blood. The inventors have now further discoveredthat AAA-GUS possessed improved functional activity to tissues of theCNS, when administered to a patient with MPS VII.

The addition of a short amino acidic peptide attached to the N-terminusof GUS or other physiology active proteins possessing CNS therapeuticactivity will endow these molecules with enhanced therapeutic benefitsfor the treatment for patients with CNS disorders. Compared with nativephysiologically active GUS, the present invention described aboveprovides a physiologically active fusion protein with increasedstability in the blood and increased therapeutic effects on the brainwhen administered to a patient with MPS VII.

Therefore, an object of this invention is 1) a polypeptide therapeuticagent with increased benefits for the CNS, 2) a method of increasingbeneficial effects on the CNS, of a protein or polypeptide possessingCNS therapeutic activity, by attaching a 4-15 acid amino acid leaderthrough chemical modification or genetic engineering of a fusion proteinand 3) a method of treatment for patients suffering from CNS relateddiseases with the afore mentioned preparation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: is a schematic diagram illustrating pCXN vector and the cloningsite in the vector for the cDNA encoding native GUS or the GUS fusionprotein

FIG. 2: illustrates the steps for the construction of an expressionvector for the production of the GUS and GUS fusion protein.

FIG. 3: is a graph showing the time profiles of the blood activitylevels of native GUS and GUS fusion protein after they areintravascularly administered in an equivalent amount.

FIG. 4: shows light microscopy of neocortex from native GUS and D₆-GUStreated mice.

The cortical neuron, hippocampus, and glia cell sections show areduction of storage (S) in D₆-GUS treated compared to GUS treated MPSVII mice.

DETAILED DESCRIPTION OF THE INVENTION

Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders(LSDs) caused by deficiency of the lysosomal enzymes needed to degradeglycosaminoglycans (GAGs).¹⁴ In MPS, the undegraded GAGs are stored inlysosomes and/or secreted into the blood stream^(15,16), and excreted inurine. MPS involve the deficiency of one of 11 enzymes needed for thestepwise degradation of DS, HS, KS, and/or CS.

ERT is an established means of treating MPS. However, improving bone andbrain pathology is still an unmet challenge because only a smallfraction of enzyme is delivered to bone and brain. Most of theenzyme-based drugs are delivered to major visceral organs like liver andspleen. Although some of the enzyme reaches bone marrow, only smallamounts of the enzyme go to bone or brain. The blood brain barrierprovides a formidable obstacle for enzymes to reach brain. Therefore,the improvement of bone and brain lesions is quite limited, even afterlong-term treatment with ERT. We have tested an acidicoligopeptide-based targeting system for delivery of enzymes to tissuesin murine MPS IVA and VII models. This strategy is based on tagging ashort peptide consisting of acidic amino acids (AAA) to the matureenzyme. The AAA-tagged enzyme had five to ten times prolonged bloodclearance compared with the untagged enzymes. The tagged enzyme wasdelivered effectively to bone, bone marrow, and brain in MPS VII miceand was more effective in reversing the storage pathology than theuntagged enzyme.

Others have shown therapeutic responses in brain of mouse models MPSVII, aspartylglycosaminuria and β-mannosidosis when higher doses andlonger treatment with enzyme is made possible.^(17,18,19) These resultsindicate that when therapeutic enzyme is administered over a sufficientperiod, at doses higher than those used in conventional ERT trials sucha therapeutic dose has a beneficial effect in an adult mouse. Thepresent invention allows such beneficial effects to be achieved with theadministration of less therapeutic enzyme.

Therefore, the present invention discloses 1) an enzyme with therapeuticbenefits for the CNS whereby said benefits are enhanced by theattachment of an AAA sequence, 2) a method of attaching an acidic aminoacid sequence to a therapeutic enzyme with benefits for the CNS so as toallow said benefits to be delivered to the CNS under conditions whichwould otherwise be ineffectual, 3) a method of treating a patient withan CNS related disease using the aforementioned AAA-therapeutic enzyme.

The inventors have previous disclosed AAA-GALNS which is herebyincorporated by reference.²⁰ This reference discloses a fusion proteinfor the treatment of disease, and a method of increasing the stabilityof a therapeutic protein in blood and transfer of said protein to bone.More specifically the therapeutic protein is GALNS and the disease isMorquio disease.

The inventors have also previously disclosed an AAA-GUS, described indetail bellow, and herein incorporated by reference.²¹ This referencediscloses a fusion protein AAA-GUS for the treatment of disease withimproved in vivo stability and a method for treating a patient with MPSVII.

The term “polypeptide therapeutic agent” refereed to in the presentinvention means any polypeptide, oligopeptide or protein which willbenefit a patient suffering from disease when administered to thepatient.

The term “acidic amino acid” or “AAA” referred to the present inventionmeans glutamic acid or aspartic acid. As the employment of these acidicamino acids in the present invention is for the purpose of constructingan acidic short peptide, they may be used in any arbitrary combinationincluding a simple use of one or the other of them alone forconstruction of such a short peptide. The number of the acidic aminoacids forming a short peptide is preferably 4-15, more preferably 4-12,and still more preferably 4-8.

A short peptide consisting of acidic amino acids may be directlyattached to the N-terminus of physiologically active human GUS via apeptide bond or like, or, instead, it may be attached via a linkerpeptide.

In the present invention “a linker peptide” is not an indispensablecomponent, for it is usable only for convenience in attaching a shortpeptide consisting of acidic amino acids to N-terminus ofphysiologically active GUS. Where it is used, a linker peptide may be ashort peptide consisting e.g., preferably of 15 or less, more preferablyof 10 or less, and still more preferably of 6 or less amino acids. Sucha linker that consists of a single amino acid molecule and linkingbetween the acidic short peptide and physiologically active GUS viapeptide bonds is also included in the definition of a linker peptide. Alinker peptide may be made of any one or more amino acids desired.

In the present invention, though there is no specific limitation as tothe method for attaching an acidic short peptide to physiologicallyactive GUS, it is of advantage, e.g., to form and use a transformantcell expressing the fusion protein consisting of the short peptide andphysiologically active GUS.

In the present invention “attachment” in reference to acidic amino acidsor AAA and therapeutic proteins or peptides or enzymes refers tocreation of a covalent bond either through the creation of a fusionprotein or through the use of chemical agents or manipulation to achievethe same result.

A fusion protein of the present invention may include a non-acidic aminoacid or a sequence of several (e.g., 3) non-acidic amino acids atN-terminus of the short peptide consisting of acidic amino acids.

A fusion protein of the present invention may be formulated into apharmaceutical composition containing the fusion protein dissolved ordispersed in a pharmaceutically acceptable carrier well known to thoseskilled in the art, for parenteral administration by e.g., intravenous,subcutaneous, or intramuscular injection or by intravenous dripinfusion.

For pharmaceutical compositions for parenteral administration, anyconventional additives may be used such as excipients, binders,disintegrants, dispersing agent, lubricants, diluents, absorptionenhancers, buffering agents, surfactants, solubilizing agents,preservatives, emulsifiers, isotonizers, stabilizers, solubilizers forinjection, pH adjusting agents, etc.

A fusion protein of the present invention may be used advantageously inplace of the conventional native enzyme protein in a substitutiontherapy for the treatment of MPS VII. In the treatment, the fusionprotein may be administered intravenously, subcutaneously, orintramuscularly. Doses and frequencies of administration are to bedetermined by the physician in charge in accordance with the conditionof his or her patient.

Preferred embodiments of the invention are described in the followingexamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims, which follow the examples.

EXAMPLE 1 Method for Construction of Expression Vectors

Vector pCXN had been constructed in accordance with a literature (7) andwas offered to us by Prof. Miyazaki at Osaka University. An expressionvector for native human GUS, pCXN-GUS, was constructed by using humanGUS cDNA that had been reported by Oshima et al. (8)(Accession No. ofGenBank for the Amino acid and cDNA sequence of Human GUS is BC014142.).An expression vector for human GUS to the N-terminus of which isattached (via a linker peptide) a short peptide (N-terminal bone tag:NBT) consisting of acidic amino acids (NBT-GUS), was constructedstarting with pCXN-GUS in the following manner. FIGS. 1 and 2schematically illustrate the process for construction.

Using pCXN-GUS as a template, PCR was carried out using LA-Taq (Takara)to amplify Δsig GUS cDNA (the sequence, nt 67-1956, left behind afterremoval of the sequence of nt 1-66 corresponding to a secretion signal,from the ORF region of the sequence set forth as SEQ ID NO:1) (for humanGUS without signal sequence, see SEQ ID NO:2), to the 5′-terminus ofwhich is attached an AgeI cleavage sequence. The PCR was carried outaccording to the instruction for use of LA-Taq, i.e., through the cyclesconsisting of 30 seconds at 94° C., (30 seconds at 94° C., 30 seconds at60° C., and 2 minutes at 72° C.)×25, and then 3 minutes at 72° C., withprimer 1 (SEQ ID NO:3), and primer 2 (SEQ ID NO:4). The cDNA thusamplified was inserted into pT7Blue vector (Novagen) to constructpT7-Δsig GUS.

The N-terminal bone tag (NBT) cDNA to be attached to the 5′-terminusthen was constructed by PCR using LA-Taq (Takara). Briefly, primer 3(SEQ ID NO:5) and primer 4 (SEQ ID NO:6) were used for the constructionof NBT-E6 cDNA, primer 5 (SEQ ID NO:7) and primer 4 (SEQ ID NO:6) forthe construction of NBT-E8 cDNA, primer 6 (SEQ ID NO:8) and primer 4(SEQ ID NO:6) for the construction of NBT-D6 cDNA, and primer 7 (SEQ IDNO:9) and primer 4 (SEQ ID NO:6) for the construction of NBT-D8 cDNA. Inthe names of the NBT cDNAs, “E6” or “E8” indicate that the NBT is madeup of 6 or 8 serially connected glutamic acid residues, respectively.Likewise, “D6” or “D8” indicates that the NBT is made up of 6 or 8connected aspartic acid residues, respectively.

Employing each pair of the above primers, which contained a portioncomplementary to each other, PCR was carried out through the cyclesconsisting of 30 seconds at 94° C., (30 seconds at 94° C., 30 seconds at60° C., 30 seconds at 72° C.)×20 minutes, and then one minute at 72° C.The thus amplified DNA fragments were inserted into pT7Blue vector(Novagen) to construct pT7-NBTs.

A human GUS cDNA recovered as a fragment of pT7 pT7-Δsig GUS cleavedwith AgeI and XbaI was inserted into the AgeI-XbaI site of pT7-NBTs toconstruct pT7-NBT-GUSs.

Then each of pT7-NBT-GUSs was cleaved with BclI, blunt-ended with T4 DNApolymerase, and cleaved with XbaI to recover NBT-GUS cDNAs.

pST-RAP-GUSB (a vector comprising the p97 signal sequence, provided byTomatsu at Saint Louis University) was cleaved with BamHI and XbaI, intowhich then was inserted the NBT-GUS cDNAs recovered above to constructpST-p97-NBT-GUSs.

pST-p97-NBT-GUSs were cleaved with EcoRI to recover respectivep97-NBT-GUS cDNAs, each of which then was inserted into the EcoRI siteof pCXN to construct a NBT-GUS expression vector, pCXN-p97-NBT-GUS. TheDNA sequence of the expression vectors' region corresponding to thep97-NBT-D6-GUS, p97-NBT-D8-GUS, p97-NBT-E6-GUS and p97-NBT-E8-GUS cDNAsare shown in the Sequence Listing (SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16,) along with their corresponding amino acidsequences (SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17),respectively.

SEQ ID NO:10 shows part of the sequence containing the NBT-E6-GUS cDNAof pCXN-p97-NBT-E6-GUS. Its nt 1-57 encode the p97 signal sequence, nt61-78 a poly Glu, nt 79-96 a linker sequence, and nt 97-1983 GUS withoutthe signal sequence.

SEQ ID NO:11 shows the NBT-E6-GUS amino acid sequence with the p97signal sequence. Aa 1-19: p97 signal sequence, aa 21-26: poly Glu, aa27-32: linker sequence, aa 33-661: GUS without signal sequence.

SEQ ID NO:12 shows part of the sequence containing the NBT-E8-GUS cDNAof pCXN-p97-NBT-E8-GUS. Its nt 1-57 encode the p97 signal sequence, nt61-84 a poly Glu, nt 85-102 a linker sequence, and nt 103-1989 GUSwithout the signal sequence.

SEQ ID NO:13 shows the NBT-E8-GUS amino acid sequence with attached p97signal sequence. Aa 1-19: p97 signal sequence, aa 21-28: poly Glu, aa29-34: linker sequence, aa 35-663: GUS without signal sequence.

SEQ ID NO:14 shows part of the sequence containing the NBT-D6-GUS cDNAof pCXN-p97-NBT-D6-GUS. Its nt 1-57 encode the p97 signal sequence, nt61-78 a poly Asp, nt 79-96 a linker sequence, and nt 97-1983 GUS withoutthe signal sequence.

SEQ ID NO:15 shows the NBT-D6-GUS amino acid sequence with attached p97signal sequence. Aa 1-19: p97 signal sequence, aa 21-26: poly Asp, aa27-32: linker sequence, aa 33-661: GUS without signal sequence.

SEQ ID NO:16 shows part of the sequence containing the NBT-D8-GUS cDNAof pCXN-p97-NBT-D8-GUS. Its nt 1-57 encode the p97 signal sequence, nt61-84 a poly Asp, nt 85-102 a linker sequence, and nt 103-1989 GUSwithout the signal sequence.

SEQ ID NO:17 shows the NBT-D8-GUS amino acid sequence with attached p97signal sequence. Aa 1-19: p97 signal sequence, aa 21-28: poly Asp, aa29-34: linker sequence, aa 35-663: GUS without signal sequence.

The proteins set forth as SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 andSEQ ID NO:17 contain the p97 secretion signal sequence. The signalsequence is removed during secretion process from the cell and thefusion proteins are thus recovered as NBT-GUS in the medium.

p97 is a cell-surface glycoprotein occurring in most human melanomas andits signal sequence consists of 19 amino acids(9). The aforementionedpCXN-p97-NBT-GUSs containing the cDNA encoding this signal sequence mayalso be constructed by the following method. Briefly, a cDNA containingthe p97 signal sequence is synthesized through the process of primersannealing and PCR amplification. LA-Taq is used as an enzyme for PCR. Asprimers having mutually complementary portions, primer 8 (SEQ ID NO:18)and primer 9 (SEQ ID NO:19) are used. PCR is performed through thecycles of 30 seconds at 94° C., (30 seconds at 94° C., 30 seconds at 60°C., 30 seconds at 72° C.)×20, and one minute at 72° C. The amplifiedcDNA containing the p97 signal sequence is cleaved with BamHI and EcoRI.Into the pCXN vector, after cleaved with EcoRI, are simultaneouslyincorporated the aforementioned NBT-GUSs cDNA recovered after the enzymetreatment and cDNA for the p97 signal sequence, givingpCXN-p97-NBT-GUSs.

SEQ ID No:18 is a forward primer, in which nt 1-5 comprise a randomsynthetic sequence, and nt 6-52 comprise part of the sequence encodingthe p97 signal.

SEQ ID No:19 is a reverse primer, in which nt 1-6 comprise a randomsynthetic sequence, and nt 7-52 comprise part of the sequence encodingthe p97 signal.

Establishment of Expression Cells

Nunclon delta-MultiDish 6 Well was inoculated with CHO-K1 cells. Afteran overnight culture in DMEM/F12/FBS medium [DMEM/F12 medium(Gibco)supplemented with 10% fetal bovine serum (Thermo Trace)], each of theexpression vector constructed above was introduced into the cells usingLipofectamine 2000 reagent. For experimental procedures, the instructionmanual attached to the Lipofectamine 2000 reagent was followed. After atwo-day incubation at 37° C. in 5% CO₂, the cells were added to 75-cm²tissue culture flasks (Iwaki) and incubated until colonies of resistantcells were formed with Genetcin (Gibco) added to the DMEM/F12/FBS mediumat the final concentration of 1 mg/mL. After formation of colonies wasconfirmed under a microscope, cells which exhibited stable expressionwere cloned by the limiting dilution-culture method. Screening forexpression cells were performed by GUS-specific enzyme activity assay ofthe culture supernatants. Cell lines thus established were subculturedin DMEM/F12/FBS medium supplemented with 0.2 mg/mL Geneticin.

Method for Measurement of GUS-Specific Enzyme Activity

After intravenous administration of native-or NBT-GUS to mice, GUSactivity in the blood was determined as follows. Briefly, 12.5 uL ofplasma sample from the mice was added to 50 uL of a solution of 10 mM4-methylumbelliferyl-β-D-glucuronide (Sigma Chemical Co., St. Louis,Mo., cat #M9130) which had been prepared using determination buffer(0.1M sodium acetate buffer pH 4.8), and reaction was allowed for 1 hrat 37° C. Then, 950 uL of stop buffer (1 M Glycine-HCl, pH 10.5) wasadded and mixed to stop the enzyme reaction. Samples of the reactionmixture were transferred to a fluorometer for measurement withexcitation 366 nm/emission 450 nm.

Expression and Purification of Native GUS and GUS Fusion Protein

Native GUS and GUS fusion proteins were produced in overexpressing CHOcells, which were grown to confluency and fed with low-serum medium(Waymouth's MB 752/1 medium, supplemented with 2% FBS/1.2 mM glutamine/1mM pyruvate) (Gibco) for purification every 24 hr. The media of theculture were pooled, centrifuged at 5,000×g for 20 min at 4° C., andfrozen at −20° C. Purification was performed using affinitychromatography (10). Briefly, the conditioned medium from cellsoverexpressing the Native GUS or a GUS fusion protein was filtered, andNaCl was added to the medium at the final concentration of 0.5 M. Themedium was applied to a 5 ml column of Affi-Gel 10 (BioRad) whichcarried an anti-human GUS monoclonal antibody and had beenpre-equilibrated with wash buffer. The column was washed at 36 mL/hourwith 20-column volumes of wash buffer. The column was eluted at 36mL/hour with 50 ml of 10 mM sodium phosphate (pH 5.0) containing 3.5 MMgCl₂. Fractions were collected and subjected to GUS activity assay.Fractions containing the enzyme activity were pooled for each of theNative or fusion proteins, diluted with an equal volume of P6 buffer (25mM Tris, pH 7.5/1 mM β-glycerol phosphate/0.15 mM NaCl/0.025% sodiumazide), and desalted over a BioGel P6 column (BioRad) pre-equilibratedwith P6 buffer. Fractions containing GUS activity were pooled, and thefinally purified active protein was stored at −80° C.

EXAMPLE 2 Stability in the Blood

Per 1 g of body weight, 1,000 U of native GUS or one of the NBT-GUSs,both purified, were administered to male, 4-month old C57BL mice (3animals/group) in the tail vein. Samples of venous blood were collectedat 2 min, 5 min, 10 min, 20 min, 30 min, 1 hr, 2 hr, 6 hr, 24 hr afterthe administration, and GUS activity in the serum was measured. Theresults are shown in FIG. 3. Comparison between theNBT-GUSs-administered groups and the native GUS-administered groupreveales that, at 2 min after the administration, the enzyme activity inthe blood was 2-fold higher in the NBT-GUSs-administered groups ascompared with the native GUS-administered group. While the enzymeactivity in the blood at 30 min after the administration was almostdisappeared in the native GUS-administered group, theNBT-GUSs-administered groups retained activity levels, which were evenhigher than the activity level found at 2 min in the nativeGUS-administered group. Afterwards, the NBT-GUSs-administered groupscontinued to show remarkably slower reduction in the enzyme activitylevels in the blood as compared with those found in the nativeGUS-administered group. Even 24 hr (1440 min) after the administration,the residual enzyme activity was detectable in the NBT-GUSs-administeredgroup. A half-life time of the enzyme activity in blood in the nativeGUS-administered group was 4.9 min, while a half-life time in blood inthe NBT-GUS-administered group was prolonged 5-6 times. The resultsdemonstrate that the stability of GUS in the body is remarkablyincreased by attaching a short peptide of acidic amino acids to theN-terminus of native GUS.

EXAMPLE 3 Effects of GUS on Brain Tissue

To compare the effectiveness of AAA-tagged and untagged GUS at clearingstorage from affected tissues in the MPS VII mouse, the inventors used aprotocol in which enzyme was given in 12 weekly treatments with 1 mg/kgenzyme. There were notable differences in which the D₆-GUS appeared tobe more effective in clearing the storage material. The parietalneocortical neurons and glia had less storage in the D₆-GUS-treated MPSVII mice. In brain, the AAA-tagged enzyme showed improved clearance ofstorage from parietal neocortical neurons and glial cells, where asstorage showed minimal or no clearance response to untagged enzyme atthe same dose. FIG. 4 shows light microscopy of tissues from native GUSand D₆-GUS treated MPS VII mice. The cortical neuron, hippocampus, andglia cell sections show a reduction of storage (S) in D₆-GUS treatedcompared to GUS treated mice.

In these studies, MPS VII/E540A^(tg) mice were used.²² These mice carrya GUS transgene that encodes an inactive enzyme, which confersimmunotolerance to the human protein. To define the clearance from theblood circulation, 1,000 units per g of body weight of D6-GUS, D8-GUS oruntagged GUS were administered to 4-month-old MPS VII mice (3animals/group) via the tail vein. Samples of venous blood were collectedat 2 min, 5 min, 10 min, 20 min, 30 min, 1 h, 2 h, 6 h, and 24 h afteradministration, and GUS activity in the serum was measured.

To determine the effectiveness of D6-GUS, D8-GUS, and untagged enzyme atreversing storage pathology, three adult animals in each group receivedtwelve weekly doses (5,000 units/g) of D6-GUS, D8-GUS, untagged enzymeor PBS by injection in the lateral tail vein.

Animals were killed 1 week after the 12th injection, and the organs wereremoved for histopathology analysis with light or electron microscopy.

For morphological evaluation, liver, spleen, kidney, brain, heart,femur, and bone marrow from 4-5 month old MPS VII mice treated withD6-GUS (n=2), D8-GUS (n=3), and untagged enzyme (n=3), or buffer (n=2)were collected at necropsy, immersion-fixed in 4% paraformaldehyde/2%glutaraldehyde in PBS, postfixed in osmium tetroxide, and embedded inSpurr's resin. For evaluation of lysosomal storage by light microscopy,toluidine blue-stained 0.5-μm-thick sections were examined. One mousetreated by D6-GUS died immediately after the 12th weekly infusion andwas not evaluated morphologically. Tissues from the treated anduntreated mice were evaluated for reduction in storage without knowledgeof their treatment. Two pathologists (CV, BL) independently evaluatedthe brain for lysosomal storage.

Some individual elements of the inventors' methodology are generallyknown or described in detail in numerous laboratory protocols, one ofwhich is Molecular Cloning 2nd edition, (1989) Sambrook, J., Fritsch, E.F. and Maniatis, J., Cold Spring Harbor. As such detailed discussion oftheir composition and methodology is superfluous.

REFERENCES

Applicants make no statement, inferred or direct, regarding the statusof the following references as prior art. Applicants reserve the rightto challenge the veracity of any statements made in these references,which are incorporated herein by reference.

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1. A polypeptide therapeutic agent with increased central nervous systemtherapeutic activity comprised of a) a physiologically active proteinwith therapeutic benefits for the central nervous system, and b) a shortpeptide which consisting of 4-15 acidic amino acids attached to thephysiology active protein via the N-terminus thereof.
 2. A polypeptidetherapeutic agent as in claim 1 wherein said physiologically activeprotein is an enzyme.
 3. A polypeptide therapeutic agent as in claim 1wherein said physiologically active protein is an enzyme known to betherapeutic in treatment of lysosomal storage disease.
 4. A polypeptidetherapeutic agent as in claim 1 wherein said physiologically activeprotein is human β-glucuronidase.
 5. A polypeptide therapeutic agent asin claim 1 wherein attaching said acid amino acid sequence of 4-15 aminoacids to the N terminus of said polypeptide increases clearance time inthe blood.
 6. A polypeptide therapeutic agent as in claim 1 wherebyattached comprises produced a fusion protein through geneticengineering.
 7. A polypeptide therapeutic agent as in claim 1 wherebyattached comprises chemically linking at least two molecules.
 8. Apolypeptide therapeutic agent as in claim 1 whereby attached is via alinker peptide.
 9. A method of increasing therapeutic benefits of aphysiologically active protein on the central nervous system wherein themethod comprises, a) a physiologically active protein with therapeuticbenefits for the central nervous system, and b) a short peptideconsisting of 4-15 acidic amino acids, which is c) attached to saidphysiology active protein via the N-terminus thereof.
 10. A method ofincreasing therapeutic benefits as in claim 9 whereby saidphysiologically active protein is an enzyme.
 11. A method of increasingtherapeutic effects as in claim 9 whereby said physiologically activeprotein is an enzyme known to be therapeutic in treatment of lysosomalstorage disease.
 12. A method of increasing therapeutic effects as inclaim 9 whereby said physiologically active protein is humanβ-glucuronidase.
 13. A method of increasing therapeutic effects as inclaim 9 whereby attaching said acid amino acid sequence of 4-15 aminoacids to the N terminus of said polypeptide increases clearance time inthe blood.
 14. A method of increasing therapeutic effects as in claim 9whereby attaching comprises a fusion protein produced through geneticengineering.
 15. A method of increasing therapeutic effects as in claim9 whereby attaching comprises chemically linking at least two molecules.16. A method of increasing therapeutic effects as in claim 9 wherebyattaching is via a linker peptide.
 17. A method of treating a patientwith a central nervous system disease by administering an effectiveamount of a physiologically active protein with therapeutic benefits forthe central nervous system, and b) a short peptide which consists of4-15 acidic amino acids, which is c) attached to the physiology activeprotein on the N-terminus thereof.
 18. A method as in claim 17 wherebysaid physiologically active protein is human β-glucuronidase.
 19. Amethod as in claim 17 whereby said central nervous system disease is alysosomal storage disease.
 20. A method as in claim 17 whereby saidcentral nervous system disease is type VII mucopolysaccharidosis.
 21. Amethod as in claim 17 whereby said physiologically active protein withsaid short peptide attached further comprises increased clearance timein the blood.